The making of a slicer: activation of human Argonaute-1.

Abstract

Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-hAgo4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 microRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad, combined with a mutation on a loop adjacent to the active site of hAgo1, results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2 without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.

(A) Overall structure of hAgo1 in complex with let-7 guide RNA. The individual domains of hAgo1 are labeled and color coded. Let-7 miRNA is shown as an orange cartoon. Nucleotides 1–10 stretch from the MID domain and pass through L2, the PIWI domain and L1. A dashed line indicates the projected path of the disordered nucleotides 11–20. Nucleotides 21 and 22 are modeled in the PAZ domain. (B) Magnified view of the path of the seed region of let-7 bound to hAgo1. The let-7 miRNA bound to hAgo1 is shown as orange sticks. The superimposed miR20a from the hAgo2-miR20a complex (PDB 4F3T) is shown as grey sticks. Structural elements are colored according to the domain colors in panel A. Residues that make kinks in the let–7 miRNA are shown as sticks. See also .

(A) Structural superposition of hAgo1 and hAgo2 based on the MID–PIWI lobe. hAgo1 is shown as a cartoon with the same color scheme as . hAgo2 is shown as a grey cartoon. (B) The same alignment in panel A but with a 90° rotation around the horizontal axis. All of the domains are colored as in panel A, but with the MID and PAZ domains deleted for clarity. Secondary structure elements in the N domain that interact with the L1, L2 and PIWI domain are indicated as are the elements composing the subdomain of the N that move in hAgo1 with respect to hAgo2. (C) Slicer assays for WT hAgo1, hAgo2, the mutants of the catalytic tetrad and chimeric hAgo2 constructs swapped with inactive hAgo1 domains. (D) Slicer assay of hAgo1 chimeras comprised of single and double domain swaps from hAgo2. (E) Plot of fraction product cleaved over a 60–minute time course showing enhanced slicing by the hAgo2 N domain. The standard error deviation from the mean of three individual replicates is plotted with data points connected by a smooth curve in Excel. (F) Slicer assay for hAgo3-hAgo2 N domain chimera. Time points were taken every 10, 30 and 60 minutes. A line designates an irrelevant lane that was cropped. Slicer assays shown in panels C, D and F are representatives of at least three individual replicates. See also .

(A) View of the superimposed active sites in the PIWI domain of hAgo1 and hAgo2. The PIWI domain of hAgo1 is colored purple and hAgo2 is grey. Conserved active site residues are labeled with yellow text. Mutations in hAgo1 that activate slicing are labeled in green. The PIWI domain loops (PL1, PL2 and PL3) that cluster near the active site are indicated. (B) Sequence alignment focused on the catalytic tetrad residues for hAgo1, hAgo2, hAgo3, Drosophila Ago1 and Ago2 (dAgo1 and dAgo2), and two other eukaryotic Argonautes for which structures are known, NcQDE-2 and KpAgo. Conserved catalytic residues are highlighted in yellow and mutations that activate hAgo1 are highlighted in green. The PIWI domain loops PL2 and PL3 are indicated. (C) Slicer assay showing activated hAgo1H by mutation of L674F. The N domain of hAgo2 enhances the slicing by the activated hAgo1. F676L mutants of hAgo2 have a severe defect in slicing. (D) Plot of fraction product cleaved over a 60–minute time course showing the activated hAgo1H L674F and the enhancement from the hAgo2 N domain. The standard error deviation from the mean of three individual replicates is plotted with data points connected by a smooth curve in Excel. (E) Mutants of the PL3 loop residues in hAgo2 show that E673 is important for slicing. (F) Mutants of F676 in hAgo2 greatly impact slicing activity. Slicer assays shown in panels C, E and F are representatives of at least three individual replicates. See also .

(A) Structure of hAgo1 with a modeled A–form duplex RNA. Domains of hAgo1 are colored as in . The PAZ domain and L1 linker are omitted for clarity. Let-7 miRNA is orange, the modeled guide RNA is red and target strand blue. The location of the scissile phosphate is indicated with scissors. The active site tetrad is shown as yellow sticks. The PL3 loop is highlighted green with important residues identified in this study shown as green sticks. (B) A close-up view of panel (A) but only the PIWI domain is shown. The PL2 and PL3 loops are indicated. (C) A–form RNA modeled in hAgo2 with the same layout as panel B. See also .